In medicine and biology, density gradients are commonly used to separate blood into distinct fractions such as red blood cells, white blood cells and plasma. The blood is overlayered onto a medium of defined density (e.g. Ficoll™, Ficoll-Paque™, GE Healthcare) and then centrifuged. During the centrifugation process, differential migration of the blood components occurs, resulting in the formation of layers of each blood fraction. Red blood cells migrate through the Ficoll layer and sediment at the bottom of the tube while white blood cells move to the interface above the Ficoll and below the plasma layers. White blood cells nominally comprise less than 1% of peripheral blood and this technique allows for their effective and facile purification.
For the effective separation of blood fractions the interface between the density gradient medium and the blood sample must be as sharp as possible, with minimal mixing. This can be difficult to achieve and a skilled, steady hand and patience are required. As such the loading of blood onto a density gradient medium prior to centrifugation is very time consuming. The interface is also fragile and if the tube is knocked the interface can easily mix and be destroyed. The removal or harvesting of the separated bands or fractions following centrifugation also poses technical challenges for the operator who must take care not to disrupt or destroy the fractions and to avoid excessive carry-over of density gradient medium.
Methods and devices have been developed to address these problems.
WO2012/149641 (Stem Cell Technologies Inc.) describes an insert for a centrifuge tube which aids density gradient separation of different cellular populations present in a sample. The insert is sized to fit into a centrifuge tube, and has a member which is positioned or stabilized within the tube by a support, typically a cylindrical support, thereby dividing the tube into a top and bottom portion. The member is typically of a concave configuration and has at least two openings, one of which is closer to the bottom end of the tube when the insert is in position and acts to allow liquid to pass through it to the bottom portion, the second acting to allow air to escape to equalize pressure. Centrifuge tubes incorporating such inserts are commercially available, i.e. SepMate™ from Stem Cell Technologies Inc.
Accuspin™ tubes, available from Sigma-Aldrich, are designed for use with density gradient medium, such as Histopaque®, for the isolation of lymphocytes and other mononuclear cells. The Accuspin tube (also known as a Leucosep™ tube available from Greiner Bio-One) is a specially designed polypropylene centrifugation tube with two chambers separated by a porous high density polyethylene barrier or frit. The density gradient medium, such as Histopaque-1077, is added to the lower chamber below the frit. The blood sample is added to the top chamber and the tube centrifuged. On centrifugation, the red blood cells sediment to the bottom of the tube containing the density gradient medium, while mononuclear cells such as lymphocytes and monocytes form a dense band at the plasma/Histopaque-1077 interface. This band can then be removed by decanting or with a pipette. Contamination with red blood cells is avoided due to the barrier between the chambers.
Floaties™ (www.Biofloaties.com) are small autoclavable polymer blend beads which are designed for the in vitro isolation of peripheral blood mononuclear cells (PBMC's) from human whole blood and cord blood samples by density gradient centrifugation. The beads are poured directly onto the top of the density gradient medium in a centrifuge tube and the blood sample gently added. The majority of the beads rise to the top of the sample which may then be centrifuged in the tube. Following centrifugation, the PBMC's form a layer or band near the plasma-density gradient medium interface, and are collected by inserting a pipette through the layer of beads.
Although a number of commercial products are available for overcoming the aforementioned problems, there is still a need for a simple and flexible means for achieving an effective separation of blood fractions in a cost effective manner. The present invention addresses this need by the provision of novel inserts, methods, centrifuge tubes and kits.